Lipid Storage Myopathies Due to Fatty Acid Oxidation Defects

Normal Pathway of Fatty Acid Oxidation

The pathway of mitochondrial FAO is outlined in Figure 40.1 . During fasting, free fatty acids are liberated from adipocytes and are transported to other tissues either as triglyceride-rich lipoproteins or bound to serum albumin. Triacylglycerols are hydrolyzed outside the cells by lipoprotein lipase to yield free fatty acids (FFAs). The mechanism of FFA entry into cells is not well understood; however, there is kinetic evidence to suggest both a saturable and nonsaturable uptake of FFAs. Once across the plasma membrane, short- (e.g. 4-carbon) and medium-chain (e.g. 8-carbon) fatty acids (less than 10 carbon atoms) are able to cross the outer and inner mitochondrial membranes as free acids to enter the mitochondrial matrix. Here they are then activated by their respective short- and medium-chain acyl-CoA synthetases to their CoA thioesters for ensuant intramitochondrial β-oxidation.

The mitochondrial membrane is impermeable to long-chain fatty acids (e.g. 16-carbon). Therefore, long-chain fatty acids diffuse or are transported to the outer mitochondrial membrane and to the endoplasmic reticulum. At both locations they are activated by conversion to their CoA thioesters. Long-chain acyl-CoA synthetase is a membrane-bound enzyme located in the endoplasmic reticulum and in the outer mitochondrial membrane. This enzyme acts on saturated fatty acids containing 10 to 18 carbon atoms and on unsaturated fatty acids containing 16 to 20 carbon atoms. Most of the activated fatty acids are directed toward mitochondrial β-oxidation. The inner mitochondrial membrane is impermeable to CoA and its derivatives, namely fatty acyl-CoA thioesters, which have been formed at the outer mitochondrial membrane. Thus these thioesters cannot directly enter the mitochondrial matrix. Therefore, the long-chain acyl-CoA must first be converted into its acylcarnitine form, e.g. palmitoylcarnitine, with release of free CoA. This is accomplished by the reversible enzyme carnitine palmitoyltransferase I (CPT I), which uses carnitine as a cofactor and is located on the inner side of the outer mitochondrial membrane. The palmitoylcarnitine is then translocated across the inner mitochondrial membrane by carnitine:acylcarnitine translocase which catalyzes a slow unidirectional diffusion of carnitine both in and out of the mitochondrial matrix in addition to a much faster mole-to-mole exchange of acylcarnitine for carnitine, carnitine for carnitine, and acylcarnitine for acylcarnitine. In the mitochondrial matrix, carnitine palmitoyltransferase II (CPT II), which is situated on the inner side of the inner mitochondrial membrane, converts palmitoylcarnitine, in the presence of free CoA, back to palmitoyl-CoA and carnitine.

There are four sequential steps in mitochondrial β-oxidation, which are composed of chain-length specific enzymes ( Figure 40.2 ). It was originally believed that the complete intramitochondrial oxidation of long-chain fatty acids required a minimum of nine enzymes including the three genetically distinct acyl-CoA dehyrogenases (short-, medium-, long-chain) and at least two enoyl-CoA hydratases (crotonase and long-chain enoyl-CoA hydratase), two L-3-hydroxyacyl-CoA dehydrogenases (short- and long-chain) and two 3-ketoacyl-CoA thiolases (acetoacetyl-CoA thiolase and generalized 3-ketoacyl-CoA thiolase). Further enzymatic and protein characterization has revealed the presence of an additional very-long-chain acyl-CoA dehydrogenase enzyme, of an acyl-CoA dehydrogenase 9 (ACAD9) that demonstrates maximal activity with unsaturated long-chain acyl-CoAs, and of a trifunctional protein which combines the activities of the long-chain enoyl-CoA hydratase, long-chain-L-3-hydroxyacyl-CoA dehydrogenase, and the long-chain thiolase enzymes. These enzymes are chain-length specific. For example, the long-chain acyl-CoA dehydrogenase has specificity for 12–18 carbon fatty acids and the medium-chain acyl-CoA dehydrogenase has specificity for 4–12 carbon fatty acids. Despite a significant overlap of substrate specificity, it appears that ACAD9 and VLCAD are unable to compensate for each other in patients with either deficiency. Studies of the tissue distribution and gene regulation of ACAD9 and VLCAD identify the presence of two independently regulated functional pathways for long-chain fat metabolism, indicating that these two enzymes are likely to be involved in different physiological functions. ACAD9 is required for the assembly of mitochondrial complex I. ACAD9 mutations result in complex I deficiency without disturbing long-chain fatty acid oxidation. This strongly contrasts with its evolutionary ancestor VLCAD, which is not required for complex I assembly and clearly plays a role in fatty acid oxidation. The trifunctional protein (TFP) is a heterocomplex of four alpha and four beta subunits, which are encoded by two nuclear genes. The alpha subunit contains the long-chain enoyl-CoA hydratase and LCHAD activities, and the beta subunit contains the long-chain 3-ketoacyl-CoA thiolase activity. There are two different biochemical phenotypes of TFP deficiency. The first includes deficiency of all three enzyme activities of the TFP, with loss of both the alpha and beta subunits by immunoblotting studies. The second has isolated LCHAD deficiency with preservation of the long-chain enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities and normal amounts of the alpha and beta subunits.

With each complete cycle, a 2-carbon fragment is cleaved and an acetyl-CoA moiety is released. In most tissues, such as muscle and heart, the acetyl-CoA is oxidized for energy production via the tricarboxylic acid cycle. In liver, about 90% of the hepatic acetyl-CoA is converted into ketones via the coordinated action of acetyl-CoA acetyltransferase, β-hydroxy β-methylglutaryl-CoA synthase, and β-hydroxy β-methylglutaryl-CoA lyase. These ketones are then exported for final oxidation by other tissues, such as brain. Ketolysis in extrahepatic mitochondria is mediated by two reversible reactions catalyzed by succinyl-CoA:3-ketoacid-CoA transferase (SCOT) and mitochondrial acetoacetyl-CoA thiolase.

The Regulation of Fatty Acid Oxidation

There are several levels of fatty acid oxidation regulation. The rate of FAO is determined by the availability of fatty acids and by the rate of utilization of β-oxidation products. The concentration of nonesterified fatty acids in plasma is regulated by the hormones glucagon, which stimulates, and insulin, which inhibits the breakdown of triacylglycerols in adipose tissue. Glucagon activates adenylate cyclase leading to an increase in the concentration of cellular cyclic AMP, which in turn activates protein kinase. One of the substrates of protein kinase in adipose tissue is the hormone-sensitive lipase. This lipase is activated by phosphorylation and inactivated by dephosphorylation. Therefore, when the concentration of glucose is low, as in fasting, there is a high glucagon-to-insulin ratio, which results in an increase in plasma nonesterified FFA. These fatty acids will enter cells, where they can be either degraded to acetyl-CoA or incorporated into other lipids. The utilization of fatty acids for either oxidation or lipid synthesis depends both on the nutritional state and on the availability of carbohydrates.

Because of its role in ketogenesis, the regulation of FAO in the liver differs and is more complex than the regulation in heart and skeletal muscle. In the fed state, the liver breaks down carbohydrates to synthesize fatty acids. In contrast, in the fasted animal, FAO, ketogenesis, and gluconeogenesis are more active. McGarry and Foster suggested that the concentration of malonyl-CoA, which is the specific reversible inhibitor of CPT I, determines the rate of FAO. In the fed state, where glucose is converted to fatty acids, the concentration of malonyl-CoA is elevated, thereby leading to an inhibition of CPT I activity. This inhibits the transfer of long-chain fatty acyl residues from CoA to carnitine so that long-chain acylcarnitines cannot be translocated into the mitochondria. Consequently, β-oxidation is depressed. When there is a change from the fed to the fasted state, hepatic metabolism shifts from glucose breakdown to gluconeogenesis, leading to a decrease in fatty acid synthesis. The concentration of malonyl-CoA decreases and the inhibition of CPT I is relieved whereby acylcarnitines are then formed and translocated into mitochondria for β-oxidation and ketogenesis. The cellular concentration of malonyl-CoA is directly related to the activity of acetyl-CoA carboxylase, which is hormonally regulated. In fasting, there is an increase in the glucagon:insulin ratio which causes an increase in cellular cAMP which, in turn, is responsible for the phosphorylation and inactivation of acetyl-CoA carboxylase. As a consequence, the concentration of malonyl-CoA and the rate of fatty acid synthesis decrease, while the rate of β-oxidation increases. A decrease in the glucagon:insulin ratio reverses these effects. Both fatty acid synthesis and FAO are regulated by the glucagon:insulin ratio. A third area that may play a key role in regulating FAO occurs in the mitochondria, where end-product inhibition of proximal β-oxidation by more distal acyl-CoA intermediates prevents excessive accumulation of acyl-CoAs even under conditions of very rapid FAO.

Alternative Pathways for Fatty Acid Metabolism

Under normal circumstances, mitochondrial β-oxidation accounts for the majority of fatty acid metabolism; however, there are other available mechanisms for the oxidation or disposal of fatty acid intermediates. Peroxisomes contain an oxidation pathway which is composed of enzymes that are genetically distinct from the mitochondrial enzymes. The peroxisome may contribute up to 20% of total cellular FAO under conditions of prolonged fasting. When there is an excessive accumulation of acyl-CoAs, they are shunted to microsomes where they undergo omega oxidation. This places a carboxyl group on the methyl-terminal end of the fatty acid and results in the formation of a dicarboxylic acid. Dicarboxylic acids (DCAs) are found in most conditions where the capacity for β-oxidation is exceeded. These include normal fasting, diabetic ketoacidosis, feeding with medium-chain triglycerides, and genetic defects in intramitochondrial β-oxidation. The specific pattern of DCAs found in serum or urine may be diagnostically useful in the identification of specific inborn errors of FAO.

Three additional mechanisms may be important when there is an impairment in mitochondrial β-oxidation. These include the conjugation of acyl groups to glycine and to carnitine and the deacylation of CoA by thioesterases. These glycine and carnitine conjugates are able to cross cellular membranes more readily than the respective CoA ester. This prevents excessive accumulation of intracellular acyl-CoAs and is important because acyl-CoA esters inhibit specific enzymes and transporters. Removal of intracellular acyl groups also preserves free CoA for other enzymes which require CoA as a cofactor. Measurement of acylcarnitines and acylglycines is diagnostically useful in the identification of genetic defects in FAO.

Central Role of Carnitine Metabolism

Carnitine (beta-hydroxy-gamma-trimethylaminobutyric acid), a water-soluble quartenary amine, has several important intracellular functions : (1) it modulates the intramitochondrial acyl-CoA/CoA sulfhydryl ratio in mammalian cells; (2) it serves as an essential cofactor for mitochondrial FAO by transferring long-chain fatty acids as acylcarnitine esters across the inner mitochondrial membrane; (3) it facilitates branched-chain alpha-keto acid oxidation; (4) it shuttles acyl moieties that have been chain-shortened by beta-oxidation out of peroxisomes in the liver; (5) it traps potentially toxic acyl-CoA metabolites that may increase in excess during acute metabolic crises through esterification to carnitine. These metabolites may secondarily impair the citric acid cycle, gluconeogenesis, the urea cycle, and fatty acid oxidation.

In omnivores, approximately 75% of carnitine sources comes from the diet with 25% from endogenous biosynthesis. The principal dietary sources include meat, poultry, fish and dairy products. Approximately 70–80% of dietary carnitine is absorbed in omnivores, whereas in strict vegetarians, endogenous carnitine synthesis provides more than 90% of total available carnitine. There are adequate carnitine concentrations in human milk and most supplemented milk-based formulas to sustain early growth and development. The plasma carnitine concentrations were found to be markedly reduced in term infants fed unsupplemented soy protein-based formulas compared to those in supplemented infants. Skeletal muscle is the major tissue reservoir of carnitine, containing over 90% of total body carnitine stores. Under normal conditions, the carnitine concentration in tissues, other than brain, is 20- to 50-fold higher than in plasma and parallels the capacity of the tissue to metabolize fatty acids; human tissue concentrations (nmol/g) are heart (3500–6000)>muscle (2000–4600)>liver (1000–1900)>brain (200–500). Thus, plasmalemmal carnitine uptake occurs across a large concentration gradient which is maintained by a transport system that is generally held to be sodium-gradient and energy-dependent. The normal serum carnitine concentration is tightly maintained by the renal threshold, which is 40 μmol/L. The kidney is capable of adjusting to wide variations in dietary carnitine as carnitine is not significantly degraded in the body. More than 90% of filtered carnitine is reabsorbed by the kidney at normal physiological plasma carnitine concentrations. Human skeletal muscle, heart, liver, kidney and brain are capable of the biosynthesis of carnitine from methionine and lysine to its immediate precursor gamma-butyrobetaine. However, the final conversion of gamma-butyrobetaine to L-carnitine by gamma-butyrobetaine hydroxylase can only be done in liver, kidney, and brain. Gamma-butyrobetaine is thus exported to these tissues for final conversion to L-carnitine. Hepatic gamma-butyrobetaine hydroxylase is developmentally regulated, being approximately 25% of adult activity at birth.

Since 1973, many patients with carnitine deficiency have been described. Originally they were divided into two groups: those with a systemic form characterized by recurrent coma with low carnitine concentrations in serum, liver and muscle versus a muscular form characterized by a progressive lipid storage myopathy in which the serum concentrations were normal and the carnitine deficiency was confined to skeletal muscle. However, with recent advances in biomedical technology, many of these cases were found to be due to intramitochondrial β-oxidation defects with secondary carnitine deficiency . For example, many cases of the systemic form were found to be due to medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. Similarly, certain cases of the myopathic form were attributed to short-chain acyl-CoA dehydrogenase (SCAD) deficiency. The secondary carnitine deficiency disorders can be divided into (1) genetic, (2) acquired, and (3) iatrogenic forms ( Box 40.1 ).

Fasting Adaptation

Fats comprise the most important and efficient fuel for oxidative metabolism. The largest reserve of fuel in the body is comprised of fatty acids stored as adipose tissue triglyceride. As liver glycogen stores are depleted within a few hours of a meal and since there are no reserve stores of protein in the body, fatty acids become the predominant substrate for oxidation quite early in fasting. In adults, approximately 80% of caloric requirements after a 24-hour period of fasting is supplied by fatty acids, which increases to 94% during more prolonged fasting. Fatty acids serve three major functions. First, the partial oxidation of fatty acids by the liver produces ketones (acetoacetate, β-hydroxybutyrate) which are an important auxiliary fuel for almost all tissues and particularly brain, as the blood-brain barrier prevents the direct use of long-chain fatty acids by the brain. This, therefore, provides an important mechanism to spare glucose oxidation and proteolysis during prolonged fasting. Second, fatty acids serve as a major fuel for cardiac and skeletal muscle. Resting muscle depends mostly on FAO. Depending upon the type, intensity, and duration of exercise, energy in working muscle is derived from either the combination of triglyceride and stored glycogen or the combination of glucose and free fatty acids. After 90 minutes, the major fuels are glucose and free fatty acids. During 1–4 hours of mild to moderate prolonged aerobic exercise, free fatty acid uptake by muscle increases by 70% and after 4 hours, free fatty acids are used twice as much as carbohydrate sources. Third, the high rates of hepatic gluconeogenesis and ureagenesis needed for maintaining fasting homeostasis are sustained by the production of ATP, reducing equivalents (nicotinamide adenine dinucleotide, reduced, +H+), and metabolic intermediates (acetyl-coenzyme A) derived from FAO.

Increased Susceptibility of the Child

Infants and young children have an increased risk of problems with fasting adaptation for several reasons. First, infants have a larger brain compared with body size which is highly dependent upon glucose and has a high rate of metabolism. Thus, infants and children show an even earlier activation of FAO with hyperketonemia within 12 to 24 hours of fasting. Second, basal energy needs in the infant are high in order to maintain body temperature, given their large ratio of surface area to mass. Their body temperature is maintained by shivering thermogenesis, which is highly dependent upon efficient FAO. Third, there is a lower activity of several key enzymes involved in energy production in the infant compared to the older child and adult, which leads to further impairment of the infant's ability to maintain glucose homeostasis.

Common Features of Fatty Acid Oxidation Disorders

It has been suggested that there are at least four clinical and laboratory features that should lead the clinician to suspect a genetic defect in fatty acid metabolism ( Box 40.2 ). These common features include: (1) acute metabolic decompensation in association with fasting; (2) chronic involvement of tissues highly dependent upon efficient fatty acid oxidation (e.g. muscle, heart, liver); (3) recurrent episodes of hypoketotic hypoglycemia; (4) alterations in the quantity of total carnitine or in the percentage of esterified carnitine in plasma and tissue.